DESCRIPTION (Applicant's description) It is increasingly apparent that both the normal, differentiated state, and the manifestation of the malignant phenotype in the epithelia, are dependent on the nature and the integrity of the surrounding stroma and the extracellu- lar matrix (ECM). While these phenomena are relatively well studied in animal models, the definition and the role of the "microenvironment" in the human tissues has lagged behind. My coworkers and I have established culture systems for the cultivation of functional epithelial cells from the rodent mammary gland in which they are able to recreate "lactating" alveoli in the culture dish. As such, we have unraveled some unexpected aspects of microenvironmental control. Together with the Co-Principal Investigators, we have applied these methods to establish a rapid and versatile assay for distinguishing normal and malignant human breast epithelial cells in a 3- dimensional organotypic assay. Our main collaborator (OWP) and his coworkers have isolated and characterized one of a very few "diploid" human cell lines (HMT3522) that in our 3-D assay behaves like normal cells from reduction mammoplasties. During passage in culture, these cells become aneuploid and after 205 passages become tumorigenic in the nude mice, and at this stage behave like the carcinomas in our assay. Passages of this line, as well as primary luminal cells from reduction mammoplasty, will be used for functional studies outlined here. We will choose 5 passages between early, intermediate and late stages of HMT3522, with the first being "normal" by the criteria of our assay, and the last, malignant by the criteria of tumor formation in the nude mice. The aims are to understand, in a well defined culture model which mimics the behavior or the cells in vivo, the influence of stromal cells (primary or secondary) on the function of the epithelial cells and vice versa. We have recently developed techniques for isolation of the major cell types from the human breast stroma. We will further characterize these cells and use them together with cells from primary tumors in 3-D cultures to explore the origin and the nature of the "stromal reaction". We have defined a number of markers to distinguish normal (reduction mammoplasty as well as HMT 3522) and malignant epithelial cells as a result of cell-ECM interactions. These include the loss of ability to form TDLU (terminal duct lobular units), loss of polarity (as measured by the localization of sialomucin and expression of E and P cadherins), changes in integrins, and components of the basement membrane, and importantly, the inability to growth arrest in 3-D. We will supplement the list with additional markers including known oncogenes and tumor suppressor genes reported to change in breast cancer, as well as assays for active TGFbeta and TGFalpha to generate an informative panel of markers. These will be used to measure gene expression and to monitor the progression to tumorigenicity after different combinations of "homotypic and heterotypic" interactions between the stromal and the epithelial cells. The long range goals are a) to define, in a physiological microenvironment, accurate and early markers for changes to the malignant phenotype as a result of stromal- epithelial interactions and b) to pave the way to understanding the detailed mechanism by which the microenvironment regulates function in normal and malignant breast.